Bottom Line:
Pulse-chase studies demonstrated a precursor-product relationship between APPFL and APPbeta as well as intracellular and secreted APPbeta fragments.In addition, trypsin digestion of intact NT2N cells at 4 degrees C did not abolish APPbeta recovered from the cell lysates.Significantly, production of APPbeta occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15 degrees C, or by expression of exogenous APP bearing the dilysine ER retrieval motif.

Affiliation: Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

ABSTRACTPrevious studies have demonstrated that NT2N neurons derived from a human embryonal carcinoma cell line (NT2) constitutively process the endogenous wild-type beta-amyloid precursor protein (APP) to amyloid beta peptide in an intracellular compartment. These studies indicate that other proteolytic fragments generated by intracellular processing must also be present in these cells. Here we show that the NH2-terminal fragment of APP generated by beta-secretase cleavage (APPbeta) is indeed produced from the endogenous full length APP (APPFL). Pulse-chase studies demonstrated a precursor-product relationship between APPFL and APPbeta as well as intracellular and secreted APPbeta fragments. In addition, trypsin digestion of intact NT2N cells at 4 degrees C did not abolish APPbeta recovered from the cell lysates. Furthermore, the production of intracellular APPbeta from wild-type APP appears to be a unique characteristic of postmitotic neurons, since intracellular APPbeta was not detected in several non-neuronal cell lines. Significantly, production of APPbeta occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15 degrees C, or by expression of exogenous APP bearing the dilysine ER retrieval motif.

Figure 8: APPβ generated in the presence of BFA is partially glycosylated. Cultures of NT2N neurons were metabolically labeled as in Fig. 7 in the presence or absence of 20 μg/ml BFA. The cell lysates were then immunoprecipitated with the antibody 53. (A) Samples in lanes 3 and 4 were treated with Nglyc F for 16 h to remove N-linked sugars, whereas immunoprecipitates in lanes 1 and 2 were treated with the vehicle. (B) Samples in lanes 2 and 4 were deglycosylated with a combination of Nglyc F, neuraminidase, and O-glycosidase for 16 h to remove both N- and O-linked chains (lanes 2 and 4); lanes 1 and 3 represent samples that were mock digested.

Mentions:
We sought next to determine if incomplete maturation of APP is indeed the cause of the shift in electrophoretic mobility of the APPβ fragment generated in the presence of BFA. Therefore, NT2N cells were metabolically labeled with [35S]methionine in the presence or absence of BFA, and APPβ immunoprecipitated from the cell lysate was incubated with N-glycosidase F (Nglyc F), an enzyme that removes N-linked carbohydrate chains. As shown, APPβ from BFA-treated NT2N neurons (Fig. 8 a, lane 1) migrated more quickly than APPβ recovered from untreated cells (Fig. 8 a, lane 2). After digestion with Nglyc F, APPβ demonstrated a mobility downshift in SDS-PAGE (Fig. 8 a, compare lanes 2 and 4). However, APPβ from BFA-treated cells (Fig. 8 a, lane 3) still migrated faster than APPβ from nontreated cells (Fig. 8 a, lane 4) despite enzymatic removal of all N-linked carbohydrate chains. Thus, the increased electrophoretic mobility of APPβ in the presence of BFA cannot be accounted for solely by differences in N-linked carbohydrate processing.

Figure 8: APPβ generated in the presence of BFA is partially glycosylated. Cultures of NT2N neurons were metabolically labeled as in Fig. 7 in the presence or absence of 20 μg/ml BFA. The cell lysates were then immunoprecipitated with the antibody 53. (A) Samples in lanes 3 and 4 were treated with Nglyc F for 16 h to remove N-linked sugars, whereas immunoprecipitates in lanes 1 and 2 were treated with the vehicle. (B) Samples in lanes 2 and 4 were deglycosylated with a combination of Nglyc F, neuraminidase, and O-glycosidase for 16 h to remove both N- and O-linked chains (lanes 2 and 4); lanes 1 and 3 represent samples that were mock digested.

Mentions:
We sought next to determine if incomplete maturation of APP is indeed the cause of the shift in electrophoretic mobility of the APPβ fragment generated in the presence of BFA. Therefore, NT2N cells were metabolically labeled with [35S]methionine in the presence or absence of BFA, and APPβ immunoprecipitated from the cell lysate was incubated with N-glycosidase F (Nglyc F), an enzyme that removes N-linked carbohydrate chains. As shown, APPβ from BFA-treated NT2N neurons (Fig. 8 a, lane 1) migrated more quickly than APPβ recovered from untreated cells (Fig. 8 a, lane 2). After digestion with Nglyc F, APPβ demonstrated a mobility downshift in SDS-PAGE (Fig. 8 a, compare lanes 2 and 4). However, APPβ from BFA-treated cells (Fig. 8 a, lane 3) still migrated faster than APPβ from nontreated cells (Fig. 8 a, lane 4) despite enzymatic removal of all N-linked carbohydrate chains. Thus, the increased electrophoretic mobility of APPβ in the presence of BFA cannot be accounted for solely by differences in N-linked carbohydrate processing.

Bottom Line:
Pulse-chase studies demonstrated a precursor-product relationship between APPFL and APPbeta as well as intracellular and secreted APPbeta fragments.In addition, trypsin digestion of intact NT2N cells at 4 degrees C did not abolish APPbeta recovered from the cell lysates.Significantly, production of APPbeta occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15 degrees C, or by expression of exogenous APP bearing the dilysine ER retrieval motif.

Affiliation:
Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.

ABSTRACTPrevious studies have demonstrated that NT2N neurons derived from a human embryonal carcinoma cell line (NT2) constitutively process the endogenous wild-type beta-amyloid precursor protein (APP) to amyloid beta peptide in an intracellular compartment. These studies indicate that other proteolytic fragments generated by intracellular processing must also be present in these cells. Here we show that the NH2-terminal fragment of APP generated by beta-secretase cleavage (APPbeta) is indeed produced from the endogenous full length APP (APPFL). Pulse-chase studies demonstrated a precursor-product relationship between APPFL and APPbeta as well as intracellular and secreted APPbeta fragments. In addition, trypsin digestion of intact NT2N cells at 4 degrees C did not abolish APPbeta recovered from the cell lysates. Furthermore, the production of intracellular APPbeta from wild-type APP appears to be a unique characteristic of postmitotic neurons, since intracellular APPbeta was not detected in several non-neuronal cell lines. Significantly, production of APPbeta occurred even when APP was retained in the ER/ intermediate compartment by inhibition with brefeldin A, incubation at 15 degrees C, or by expression of exogenous APP bearing the dilysine ER retrieval motif.